Mixed mode chromatography involves the use of solid phase chromatographic supports that employ multiple chemical mechanisms to adsorb proteins or other solutes. Examples include but are not limited to chromatographic supports that exploit combinations of two or more of the following mechanisms: anion exchange, cation exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding, pi-pi bonding, and metal affinity.
Mixed mode chromatography supports provide unique selectivities that cannot be reproduced by single mode chromatography methods such as ion exchange, however method development is complicated, unpredictable, and may require extensive resources. Even then, development of useful procedures may require long periods of time, as exemplified by hydroxyapatite.
Hydroxyapatite is a crystalline mineral of calcium phosphate with a structural formula of Ca10(PO4)6(OH)2. Chemically reactive sites include pairs of positively charged calcium atoms and triplets of negatively charged phosphate groups. The interactions between hydroxyapatite and proteins are multi-modal, hence its classification as a mixed mode support. One mode of interaction involves metal affinity of protein carboxyl clusters for crystal calcium atoms. Another mode of interaction involves cation exchange of positively charged protein amino residues with negatively charged crystal phosphates (Gorbunoff, Analytical Biochemistry 136 425 (1984); Kawasaki, J., Chromatography 152 361 (1985)).
The individual contributions of the two mechanisms to the binding and elution of a particular protein can be controlled in part by the choice of salts used for elution. The cation exchange interaction can be controlled with a gradient of any salt, including phosphate salts, sulfates, nitrates, or chlorides, specifically including sodium chloride and potassium chloride. The calcium affinity mode is inert to most commonly used non-phosphate salts. Thus proteins that bind by interaction with the calcium groups on hydroxyapatite cannot be eluted by sodium chloride alone. They can be eluted with phosphate salts.
Hydroxyapatite is commonly used for purification of antibodies, especially from partially purified preparations. The column is usually equilibrated and the sample applied in a buffer that contains a low concentration of phosphate. Adsorbed antibodies are often eluted in an increasing gradient of phosphate salts (Gagnon, Purification Tools for Monoclonal Antibodies, Chapter 5, Validated Biosystems, Tucson, ISBN 0-9653515-9-9 (1996); Luellau et al., Chromatography 796-165 (1998)). Gradients of phosphate combined with non-phosphate salts such as sodium chloride have also been used for protein purification, including antibody purification (Freitag, “Purification of a recombinant therapeutic antibody by hydroxyapatite chromatography,” Oral presentation, 2d International Hydroxyapatite Conference, San Francisco (2001)). One such approach involves the application of a gradient of sodium chloride or potassium chloride while a low level of phosphate is held constant (Kawasaki et al., Eur. J. Biochem., 155-249 (1986); Sun, “Removal of high molecular weight aggregates from an antibody preparation using ceramic hydroxyapatite,” Oral presentation, 3rd International Hydroxyapatite Conference, Lisbon (2003); Gagnon et al., “Practical issues in the use of hydroxyapatite for industrial applications,” Poster BIOT 322, 232nd meeting of the American Chemical Society, San Francisco, (2006) [http://www.validated.com/revalbio/pdffiles/ACS_CHT 0—02.pdf]; Wyeth et al., U.S. Patent Application, Publication No. WO/2005/044856 (2005)). This approach has also been applied to antibody purification with fluorapatite (Gagnon et al., “Simultaneous removal of aggregate, leached protein A, endotoxin, and DNA from protein A purified monoclonal IgG with ceramic hydroxyapatite and ceramic fluorapatite,” Oral Presentation, Wilbio Conference on Purification of Biological Products, Santa Monica, (2005) [http://www.validated.com/revalbio/pdffiles/PBP—2005.pdf]). Fluorapatite is prepared by fluoridating hydroxyapatite. This substitutes fluoride for the hydroxyl groups creating a mineral with the structural formula Ca10(PO4)6F2.
Hydroxyapatite has been shown to yield a high degree of purification in a single step. However, the presence of phosphate and other ions may reduce binding capacity to a degree that makes either hydroxyapatite or fluorapatite economically unsuitable as capture methods (Gagnon et al., Hydroxyapatite as a Capture Method for Purification of Monoclonal Antibodies, IBC World Conference and Exposition, San Francisco (2006) [http://www.validated.com/revalbio/pdffiles/Gagnon_IBCSF06.pdf]). This prevents them from being competitive with capture methods that are relatively unaffected by phosphate and salt concentration, such as protein A affinity chromatography.
Most non-antibody protein contaminants elute before antibodies, but antibodies from different clones elute in different areas of the elution profile and may therefore overlap to varying degrees with contaminating proteins. Known methods for enhancing the separation are often ineffective and may be undesirable for economic reasons as well. For example, a shallow linear elution gradient can be applied but this has the negative side effects of increasing the buffer volume and process time, and it may still fail to achieve the desired purity.
Hydroxyapatite has been shown to be effective for removal of degraded forms of antibodies such as fragments, but selectivity is highly dependent on whether elution is conducted with a chloride gradient or with a phosphate gradient.
Hydroxyapatite and fluoroapatite have been shown to be effective for removal of aggregates from many antibody preparations. Antibody aggregates usually elute after antibodies but may coelute with antibodies to varying degrees. Aggregate removal is important because aggregates are known to contribute to nonspecific interactions that reduce the shelf stability, sensitivity, accuracy, and reproducibility of analytical results in conjunction with in vitro diagnostic applications. Aggregates are known to mediate adverse pharmacological effects, such as complement activation, anaphylaxis, or formation of therapy-neutralizing antibodies in conjunction with in vivo therapeutic applications. Aggregates also reduce purification efficiency by requiring additional steps to achieve adequately low aggregate levels in the final product. Elution of hydroxyapatite and fluorapatite with chloride gradients at low fixed concentrations of phosphate has been shown to be more effective than simple phosphate gradients, but even this approach may not be sufficient for all antibody preparations.
Various other mixed mode chromatography methods for antibody purification have been introduced in recent years. Examples of commercial products exploiting mixed mode functionalities include but are not limited to MEP Hypercel (Pall Corporation); Capto-MMC, Capto-Adhere, Capto-Q, Capto-S (GE Healthcare); and ABx (J.T. Baker). These products have varying degrees of ability to remove aggregates, host cell proteins, DNA, and virus from antibody preparations, but as with hydroxyapatite, method development is complex and unpredictable, and their utility as capture methods is often limited by low capacity.
Aqueous-soluble nonionic organic polymers are known in the field of protein purification for their ability to precipitate proteins, including antibodies. They have also been reported to increase the retention of proteins in protein A affinity chromatography and ion exchange chromatography (Gagnon, Purification Tools for Monoclonal Antibodies, Chapter 5, Validated Biosystems, Tucson, ISBN 0-9653515-9-9 (1996); Gagnon et al., “Multiple mechanisms for improving binding of IgG to protein A,” Poster, BioEast, Washington D.C., (1992); Gagnon et al., “A method for obtaining unique selectivities in ion exchange chromatography by adding organic solvents to the mobile phase,” Poster and Oral presentation, 15th International Symposium on HPLC of Proteins, Peptides, and Polynucleotides, Boston (1995) [http://www.validated.com/revalbio/pdffles/p3p95iec.pdf]). Such organic polymers include but are not limited to polyethylene glycol (PEG), polypropylene glycol, polyvinylpyrrolidone, dextran, cellulose, and starch, of various polymer molecular weights. PEG is an organic polymer with a structural formula of HO—(CH2—CH2—O)n—H. In addition to its applications for protein fractionation, it is known as a protein stabilizer appropriate for use in pharmaceutical formulations.